1. Field of the Invention
The present invention relates generally to the field of diagnostic compositions and, more particularly, to diagnostic tests useful in the qualitative and quantitative determination of urea in fluids, especially blood and urine.
2. Description of the Prior Art
Urea is the major end product of protein metabolism in man and other mammals. This product is formed in the liver, passes into the blood and is excreted through the kidneys into the urine, where it constitutes the major fraction of organic substances present. An excess of blood urea, referred to as azotemia, almost invariably indicates impairment of renal function. The determination of blood urea (usually expressed as urea nitrogen in the United States) is one of the most routinely performed clinical chemistry tests. Urea "clearance", or removal of urea from the blood by the kidneys, can be determined by urine urea concentration and used as a measure of glomerular filtration rate.
Routine clinical chemistry methods used to determine urea concentration in biological fluids are classified as direct, through the condensation of urea with suitable reagents able to form a measurable chromogen, or indirect, through the determination of ammonia as a product of urease action on urea.
The direct method involving diacetylmonoxime, described in R. J. Henry, et al, Clinical Chemistry--Principles and Technics, Harper & Row, 2nd Edition (1974), has been widely applied in routine clinical chemistry, both with manual and automated procedures. This method suffers many disadvantages: nonspecificity due to interfering reactions with citrulline, allantoin, and other body fluid components; the need for daily preparation of a standard curve or two point calibration; rapid loss of color due to photosensitivity; and manipulative problems such as the high temperature required, odor and irritating fumes. For these reasons, the indirect enzymatic methods are generally preferred.
Indirect methods are based on enzymatic conversion of urea to ammonia by urease (urea amidohydrolase, EC 3.5.1.5), according to the reaction: ##STR1## and subsequent determination of the liberated ammonia with suitable reagent system (R. Richterich, "Clinical Chemistry--Theory and Practice", translated from the 2nd German Edition, S. Karger, Basel (1969) and H. U. Bergmeyer, "Methods of Enzymatic Analysis", 2nd English Edition, Vol. 4, Verlag Chemie--Academic Press, New York (1974)). Specificity is the chief advantage of enzymatic methods since only urea is hydrolyzed by urease.
The most popular laboratory method is based on The Berthelot reaction in which ammonium ions react in alkaline medium with phenol and hypochlorite to give the blue dye indophenol. Urea determination by this method requires two steps: in the first step the sample is incubated at 37 degrees C. (usually from 10 to 20 minutes) with urease at the pH most suitable for the enzymatic reaction (i.e., around 6.5); in the second step alkaline solutions of phenol and hypochlorite are added and incubation is carried out for developing color, usually at 37 degrees C., from 10 to 30 minutes.
Another method is described in Bergmeyer, supra, which allows determination of ammonium ions derived from the enzymatic reaction with urease by the enzyme glutamate dehydrogenase (GlDH), according to the reaction: ##STR2## with measurement of the decrease in the reduced nicotinamide adenine dinucleotide (NADH) absorbance at 340 (or 334, or 366) nanometers (nm). Various changes have been made in this method in order to use it as a kinetic analysis with one step and one reagent preparation. In spite of these improvements, the use of this method in routine clinical chemistry remains limited because of the high cost of the reagents and lack of applicability to automated continuous-flow analyzers.
U.S. Pat. No. 4,194,063 describes a composition and method for the determination of urea in which the ammonia liberated from the urea by the action of urease is detected by condensation with a .beta.-diketone in the presence of an aldehyde and the color produced is monitored spectrophotometrically or spectrofluorimetrically. Although a single-step procedure, this method requires either a long incubation period or high reaction temperatures to perform the assay in a reasonable time.
R. J. Roon and B. Lowenberg in "Methods in Enzymology", Colowick & Kaplan Ed., Academic Press, XVII, 317 (1970) described the preparation and properties of urea amidolyase (EC 3.5.1.45). The Roon method, designed to assay the activity of urea amidolyase preparations, involves the spectrophotometric determination of adenosine diphosphate (ADP) using phosphoenolpyruvate, pyruvate kinase and lactate dehydrogenase, monitoring the decrease in absorbance resulting from the oxidation of DPHN (syn. NADH). A urea assay carried out by Roon's pathway would give no advantage over current UV methods by glutamate dehydrogenase. In fact, due to the stoichiometry of the reactions involved, this pathway would have half the sensitivity of the glutamate dehydrogenase pathway.
U.S. Pat. No. 4,246,342, assigned to Toyo Jozo, discloses an analytical method for the determination of pyruvate and suggests that ADP may be determined by the pyruvate pathway if first reacted with phosphoenol pyruvate and pyruvate kinase to generate pyruvate.
It has been found that an enzymatic urea assay based on the use of urea amidolyase can be accomplished through a pathway allowing a sensitive colorimetric one-step assay which provides improved results over prior art methods utilizing urease.